Dataset: oxidative stress TEP

Name: Experimental results: Exopolymer production by phytoplankton under oxidative stress; conducted at the Thornton lab, TAMU from 2007-2012 (Diatom EPS Production project)
Published: 2014-04-15
Keywords: oceans

Cite This Dataset

DOI:10.1575/1912/bco-dmo.511217.1

Project:

Effect of Temperature on Extracellular Polymeric Substance Production (EPS) by Diatoms (Diatom EPS Production)

Principal Investigator:

Daniel C.O. Thornton (Texas A&M University, TAMU)

BCO-DMO Data Manager:

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2014-04-15

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Experimental results: Exopolymer production by phytoplankton under oxidative stress; conducted at the Thornton lab, TAMU from 2007-2012 (Diatom EPS Production project)

Abstract:

Data from laboratory experiment on exopolymer production by the diatom Thalassiosira wessiflogii (CCMP 1051) and the cyanobacterium Synechococcus elongates_cf (CCMP 1379) under conditions of oxidative stress.

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Archival Copy

Version Date	Archive	Persistent Identifier (PID)	Date PID Issued
2014-04-15	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.1575/1912/bco-dmo.511217.1	2019-11-21

Description

Data from laboratory experiment on exopolymer production by the diatom Thalassiosira wessiflogii (CCMP 1051) and the cyanobacterium Synechococcus elongates_cf (CCMP 1379) under conditions of oxidative stress.

Related references:
Chen, J. 2014. Factors affecting carbohydrate production and the formation of transparent exopolymer particles (TEP) by diatoms. Ph.D. dissertation, Texas A&M University, College Station, TX.

Methods & Sampling

Growth of the phytoplankton
The diatom Thalassiosira wessiflogii (CCMP 1051) and the cyanobacterium Synechococcus elongates_cf (CCMP 1379) were obtained from the National Center for Culture of Marine Algae and Microbiota (NCMA). Replicated (n = 3) Batch cultures were grown in artificial seawater (Berges et al. 2001) containing nitrogen, phosphorus and silicon at 400 µM (as NaNO3), 25 µM (NaH2PO4), and 400 µM (Na2SiO3), respectively. Culture temperatures were maintained at 20 ± 1 °C. Photon flux density on the surface of the culture bottles was 40 to 45 µmol m-2 s-1 on a 14 hour light: 10 hour dark cycle. During exponential growth, each culture was split into three treatments in which oxidative stress was induced by the addition of hydrogen peroxide at final concentrations of 0 (control), 10 and 100 µM H2O2. The treatments were sampled once a day over the next three days.

Measures of phytoplankton abundance and biomass
Counts of 400 cells from each culture were made using hemocytometers (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984) using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Turbidity of the cultures, used as an indicator of growth, was measured by absorbance at 750 nm in a 1 cm path cuvette using a UV-Mini 1240 spectrophotometer (Shimadzu Corporation).

Chlorophyll a concentration 90% acetone extractions from biomass retained on GF/C (Whatman) were measured using a Turner Designs 700 fluorometer, which was calibrated using chlorophyll a standards (Sigma) (Arar and Collins 1997). The extract was diluted with 90% acetone if the chl a concentration were too high.

Cell permeability
Uptake and staining with the membrane-impermeable SYTOX Green (Invitrogen) was used to determine what proportion of the diatom population had permeable cell membranes (Veldhuis et al. 2001, Franklin et al. 2012). Four hundred cells were examined using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging) and the number of cells that stained with SYTOX Green was enumerated.

TEP staining and analysis
Transparent exopolymer particles (TEP) were sampled according to Alldredge et al. (1993) and TEP abundance was enumerated by image analysis (Logan et al. 1994, Engel 2009). Ten photomicrographs were taken of each slide using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Images were analyzed using ImageJ software (National Institutes of Health) based on the method of Engel (2009). Thresholding during image processing was done using the triangle method (Zack et al. 1977).

CSP staining and analysis
Coomassie staining particles (CSP) were sampled according to Long and Azam et al. (1996) and CSP abundance was enumerated by image analysis (Logan et al. 1994, Engel 2009). Ten photomicrographs were taken of each slide using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Images were analyzed using ImageJ software (National Institutes of Health) based on the method of Engel (2009). Thresholding during image processing was done using the triangle method (Zack et al. 1977).

Quantum yield of photosystem II
The quantum yield of photosystem II was used as an indicator of phytoplankton health and measured using the saturating pulse method (Genty et al. 1989) using a pulse amplitude modulated fluorometer (PAM-210, Heinz Walz GmbH) folowing a protocol based on Marwood et al. (1999).

Caspase-like activity
Caspase-like activity was measured based on the method of Bouchard & Purdie (2011). Phytoplankton were collected by centrifugation, then lysed in a buffer, and the caspase-3 like activity was measured in the extracted proteins using a Enzcheck Caspase-3 Assay Kit #1 (Invitrogen inc.). The fluorescent product was measured by fluorescence using a microplate reader (SPECTRAmax GeminiEM, Molecular Devices).

References cited
Alldredge, A. L., Passow, U. & Logan B. E. 1993. The abundance and significance of a class of large, transparent organic particles in the ocean. Deep-Sea Res. Oceanogr., I. 40: 1131-1140. doi:10.1016/0967-0637(93)90129-Q

Arar, E. J. & Collins, G. B. 1997. Method 445.0. In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae by Fluorescence U.S. Environmental Protection Agency, Cincinnati, Ohio.

Berges, J. A., Franklin D. J. & Harrison, P. J. 2001. Evolution of an artificial seawater medium: Improvements in enriched seawater, artificial water over the last two decades. J. Phycol. 37:1138-1145. doi:10.1046/j.1529-8817.2001.01052.x

Bouchard, J. N., Purdie, D. A. 2011. Effect of elevated temperature, darkness, and hydrogen peroxide treatment on oxidative stress and cell death in the bloom-forming toxic cyanobacterium Microcystis aeruginosa. J. Phycol., 47(6), 1316-1325. doi:10.1111/j.1529-8817.2011.01074.x

Engel, A. 2009. Determination of Marine Gel Particles. In Wurl, O. [Ed.] Practical Guidelines for the Analysis of Seawater. CRC Press, Taylor & Francis Group, Boca Raton, Florida, pp.125-142.

Franklin, D. J., Airs, R. L., Fernandes, M., Bell, T. G., Bongaerts, R. J., Berges, J. A. & Malin, G. 2012. Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana: Cell staining, chlorophyll alterations, and dimethylsulfoniopropionate (DMSP) metabolism. Limnol. Oceanogr. 57: 305–317. doi:10.4319/lo.2012.57.1.0305

Genty, B., Briantais, J. M., Baker N. R. 1989. The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence, Biochimica et Biophysica Acta, 990(1), 87-92. doi:10.1016/S0304-4165(89)80016-9

Guillard, R. R. L. & Sieracki, M. S. 2005. Counting cells in cultures with the light microscope. In Andersen R. A. [Ed.] Algal Culturing Techniques. Elsevier Academic Press, Burlington, MA, pp. 239-252.

Logan, B. E., Grossart, H. P. & Simon, M. 1994. Direct observation of phytoplankton, TEP and aggregates on polycarbonate filters using brightfield microscopy. J. Plankton Res.16: 1811-1815.doi:10.1093/plankt/16.12.1811

Marwood, C. A., Smith, R. E. H., Soloman, K. R., Charlton, M. N., Greenberg, B. M. 1999. Intact and photomodified polycyclic aromatic hydrocarbons inhibit photosynthesis in natural assemblages of Lake Erie phytoplankton exposed to solar radiation. Ecotox Environ Safe 44:322-327. doi:10.1006/eesa.1999.1840

Parsons, T. R., Maita, Y. & Lalli, C. M. 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford, UK.

Passow, U. & Alldredge, A. L. 1995. A dye-binding assay for the spectrophotometric measurement of transparent exopolymer particles (TEP). Limnol. Oceanogr. 40: 1326-1335. doi:10.4319/lo.1995.40.7.1326

Veldhuis, M. J. W., Kraay, G. W. & Timmermans, K. R. 2001. Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. Eur. J. Phycol. 36: 167–177. doi:10.1080/09670260110001735318

Zack, G. W., Rogers, W.E., Latt S. A. 1977. Automatic-measurement of sister chromatid exchange frequency, J. Histochem. Cytochem., 25(7), 741-753. doi:10.1177/25.7.70454

Data Processing Description

Limited processing was necessary with this dataset. As this was a laboratory experiment it was designed in such a way to ensure that the parameters measured were likely to be within a measurable range and therefore there were no measurements below detection limits. Chlorophyll concentrations were frequently too high; this was resolved by diluting the sample into the measurable range. Measured parameters were normalized to volume as most of the parameters were expressed as concentrations.

Data Files

File
oxidative_stress_TEP.csv (Comma Separated Values (.csv), 9.17 KB) MD5:67c7258f292bfea9e9fc4dd1c82ffa01 Primary data file for dataset ID 511217

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-0726369

Deployments

Deployment	Synonyms	Start Date	Platform	Investigator
lab_Thornton		2007-09-01	TAMU	Daniel C.O. Thornton (Principal Investigator)	data info

Instruments

Fluorescence Microscope

Supplied Name: Epifluorescence Microscope

Supplied Description:

Cell permeability was determined using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging).

Instrument Type

Generic Name: Fluorescence Microscope

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB06/

Generic Description:

Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments.

Fluorometer

Supplied Name: Pulse Amplitude Modulated Fluorometer

Supplied Description:

The quantum yield of photosystem II was measured using the saturating pulse method (Genty et al. 1989) using a pulse amplitude modulated fluorometer (PAM-210, Heinz Walz GmbH).

Instrument Type

Generic Name: Fluorometer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/113/

Generic Description:

A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. The instrument is designed to measure the amount of stimulated electromagnetic radiation produced by pulses of electromagnetic radiation emitted into a water sample or in situ.

Hemocytometer

Supplied Description:

Counts of 400 cells from each culture were made using hemocytometers (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984) using a light microscope.

Instrument Type

Generic Name: Hemocytometer

Generic Description:

A hemocytometer is a small glass chamber, resembling a thick microscope slide, used for determining the number of cells per unit volume of a suspension. Originally used for performing blood cell counts, a hemocytometer can be used to count a variety of cell types in the laboratory. Also spelled as "haemocytometer". Description from:
http://hlsweb.dmu.ac.uk/ahs/elearning/RITA/Haem1/Haem1.html.

Microscope - Optical

Supplied Name: Light Microscope

Supplied Description:

Counts of 400 cells from each culture were made using hemocytometers (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984) using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). A light microscope (Axioplan 2, Carl Zeiss MicroImaging) was also used to enumerate TEP and CSP by image analysis.

Instrument Type

Generic Name: Microscope - Optical

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/

Generic Description:

Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope".

Turner Designs 700 Laboratory Fluorometer

Supplied Name: Turner Designs 700 Fluorometer

Supplied Description:

Instrument Type

Generic Name: Turner Designs 700 Laboratory Fluorometer

Acronym: TD-700

Community Identifier: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0510/

Generic Description:

The TD-700 Laboratory Fluorometer is a benchtop fluorometer designed to detect fluorescence over the UV to red range. The instrument can measure concentrations of a variety of compounds, including chlorophyll-a and fluorescent dyes, and is thus suitable for a range of applications, including chlorophyll, water quality monitoring and fluorescent tracer studies. Data can be output as concentrations or raw fluorescence measurements.

UV Spectrophotometer-Shimadzu

Supplied Name: UV-Mini 1240 Spectrophotometer

Supplied Description:

Turbidity of the cultures was measured by absorbance at 750 nm in a 1 cm path cuvette using a UV-Mini 1240 Spectrophotometer (Shimadzu Corporation).

Instrument Type

Generic Name: UV Spectrophotometer-Shimadzu

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB20/

Generic Description:

The Shimadzu UV Spectrophotometer is manufactured by Shimadzu Scientific Instruments (ssi.shimadzu.com). Shimadzu manufacturers several models of spectrophotometer; refer to dataset for make/model information.

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
species	Species name.	dimensionless	species
day	Day of the experiment.	dimensionless	day
H2O2	Hydrogen peroxide concentration.	micromolar (uM)	H2O2
turbidity	Turbidity of the cultures measured by absorbance at 750 nm in a 1 cm path cuvette using a spectrophotometer.	NTU	turbidity
cell_conc	Cell concentration. Counts of 400 cells were made by transmitted light microscopy using a hemacytometer (Fuchs-Rosenthal ruling Hauser Scientific) (Guillard & Sieracki 2005).	cells per milliliter (cells mL-1)	no_bcodmo_term
chla	Chlorophyll a measured by fluorescence (Arar & Collins 1997; Method 445.0. EPA).	micrograms per liter (ug L-1)	chl_a
PSII	Quantum yield of photosystem II measured after Marwood et al. (1999) using pulse amplitude modulated chlorophyll fluorometer.	quantum yield of photosystem II	no_bcodmo_term
caspase_like_activity	Caspase-like activity was measured after Bouchard & Purdie (2011).	relative fluorescence units per milligrams protein per hour (RFU mg protein-1 h-1)	no_bcodmo_term
stained_cells_pcnt	% of SYTOX Green stained cells. Uptake and staining with the membrane-impermeable SYTOX Green (Invitrogen) was used to determine what proportion of the diatom population had permeable cell membranes (Veldhuis et al. 2001; Franklin et al. 2012). Four hundred cells were examined using an epifluorescence microscope and the number of cells that stained with SYTOX Green was enumerated.	percent (%)	no_bcodmo_term
TEP_conc	Transparent exopolymer particles (TEP) concentration. TEP retained on 0.4 polycarbonate filters and stained with Alcian blue (Alldredge et al. 1993).	micrometers TEP per milliliter (um2 mL-1)	no_bcodmo_term
TEP_abund	Transparent exopolymer particles (TEP) abundance. TEP retained on 0.4 polycarbonate filters and stained with Alcian blue (Alldredge et al. 1993).	TEP per milliliter (mL-1)	no_bcodmo_term
TEP_per_chla	Transparent exopolymer particles (TEP) per chlorophyll a.	square millimeters of TEP per nanogram of chla (mm2 (ng chl. a)-1)	no_bcodmo_term
CSP_conc	Coomassie staining particles (CSP) concentration. CSP retained on 0.4 polycarbonate filters and stained with Coomassie briliant blue blue (Long & Azam 1996).	micrometers CSP per milliliter (um2 mL-1)	no_bcodmo_term
CSP_abund	Coomassie staining particles (CSP) abundance. CSP retained on 0.4 polycarbonate filters and stained with Coomassie briliant blue blue (Long & Azam 1996).	CSP per milliliter (mL-1)	no_bcodmo_term
CSP_per_chla	Coomassie staining particles (CSP) per chlorophyll a.	square millimeters of CSP per nanogram of chlorophyll a (mm2 (ng chl. a)-1)	no_bcodmo_term